Screening of Some Native and Foreign Accessions of Spinach For

Iran Agricultural Research (2019) 38(1) 87-99

Screening of some native and foreign accessions of spinach for

Shiraz spring culture in Isfahan University R. Abolghasemi1*, M. Haghighi1, N. Etemadi1, A. Soorni2, P. Jafari3

1Department of Horticulture, College of Agriculture, Isfahan University of Technology, Isfahan, I. R. Iran 2Department of Biotechnology, College of Agriculture, Isfahan University of Technology, Isfahan. I. R. Iran 3Isfahan Agricultural and Natural Resources Research and Education Center, Isfahan, I. R. Iran

* Corresponding Author: [email protected] DOI: 10.22099/iar.2019.5317

ARTICLE INFO ABSTRACT- Spinach is one of the most important green leafy vegetables (Spinacia Article history: oleracea L.). The qualitative and quantitative traits of the spinach depends on the weather conditions. Screening the foreign accession compared with the Iranian ones is 1 Received February 2019 necessary for breeding purposes. In order to study the vegetative characteristics of 44 Accepted 26 August 2019 endemic and foreign accessions, an experiment was conducted in a randomized complete Available online 12 October 2019 block design (RCBD) with three replications and 18 observations in spring, 2018. Quantitative and qualitative parameters of spinach were evaluated based on descriptors Keywords: investigated by Bioversity International Plant Genetic Resources Institute. In general, the Breeding intensity of the green color in the leaf of foreign accessions was higher than that of the Morphologic assessment endemic spinach. In this study, the highest yield belonged to “Viroflay” (71.224 ton/ha) Viroflay accession among foreign accessions and among Iranian accessions, “Varamin88” (52.6 ton/ha) had Spinach the highest performance. Maximum and Minimum yields from 71224 to 8870 (kg/ha) Yield belonged to accessions “Viroflay” and “Virginia savoy blight”, respectively. “D’inverno” accession showed the longest period of spring growth (89.66 days) and among the Iranian accessions, “Hamadan2” (77 days), “Varamin88” (72 days) and “Varamin Prickly” (69.66 days) showed the longest growth in Isfahan environmental conditions, respectively. The highest percentage of female plants was observed in “Monatol” accession. Among the endemic accessions, “Lorestan5” showed the highest female plant percentage. Results of this study showed that Iranian accessions such as “Kashan”, “Lorestan6” and “Varamin Advanced Prickly” are suitable for mechanical harvesting due to their plant form, leaf and petiole attitude and can be used for plant breeding purposes. According to the cluster analysis, the accessions in this study were divided into two large groups (group I and group II), according to which, “Varamin88” accession was placed beside the foreign outstanding accession in cluster I. .

INTRODUCTION

Spinach (Spinacia oleracea L.) is an annual, cold- in China and the USA (Sabaghnia et al., 2014; Ebadi- tolerant plant; anemophily and dioeciously belonging to segheloo et al., 2014). The seventh rank among the Chenopodiaceae family. The genus Spinacia includes countries of the world belongs to Iran with 105 two other species including S. turkestanica ilj. and S. thousand tons spinach production per year (Sabaghnia et tetrandra Stev. They are referred to as the potential al., 2014). Kunicki et al. (2010) reported that spinach ancestors of current spinach (Andersen and Torp, 2011). yield varied from 18.6 to 44.8 tons/ha depending on the Since ancient Iranians were aware of the nutritional environmental conditions and cultivars. Spinach value of spinach, researchers maintain it likely belongs cultivation can be done in the fall and spring, but due to to Iran (Asadi and Hasandokht, 2007; Hochmuth et al., bolting in the spring, it should be cultivated 50 to 60 2003; Imani, 2013). Wild species of spinach are days before the long day and hot weather (Jafari and distributed as a vegetable in the Alborz and Zagros Jalali, 2017; Zaferaniye, 2015). Currently, there are mountains of Iran (Imani, 2013). various domestic and wild populations of spinach in Spinach is one of the most important green leafy Iran whose identification can lead to the introduction of vegetables which contains minerals, is full of iron and high-yielding populations and disease-resistant ones vitamins, and is consumed fresh or its leaves and stems (Eftekhari et al., 2010). are processed. Spinach production in the world was 22 The genetic diversity of spinach in the world has million tons in 2013, 92 percent of which was produced been studied using molecular and biochemical markers Abolghasemi et al. / Iran Agricultural Research (2019) 38(1) 87-99 and domestic researchers have studied the matters were weak, fertilization was done by releasing morphological traits of this plant (Sabaghnia et al., 2014; 40 tons/h of organic fertilizers. Each replication Kuwahara et al., 2014; Asadi and Hasandokht, 2007). In consisted of three rows with 25 cm between rows at a research studied the properties of 54 populations of around 1 cm depth and plot size was 1.75 m2. After seed spinach, the iron concentration, the yield and percentage germination and plant growth, 18 observations were of female plants were significantly different between the kept in each plot. populations (Sabaghnia et al., 2014; Ebadi-segheloo et al., 2014). In another study, Eftekhari et al. (2010) The Measured Parameters reported that “Varamin2” accession has the highest In order to evaluate the morphological characteristics, performance among the Iranian spinach. Also, in the after plant growth (35-50 days after sowing), 6 plants study of agronomic and morphological traits of autumn were randomly selected from each replicate and cultivars between 29 spinach accessions, a significant transferred to the laboratory. In the laboratory, from difference was observed between the traits (Asadi and each plant, four leaves from four directions of the plant Hasandokht 2007; Eftekhari et al., 2010). In another were selected and the following characteristics were study, researchers explained that “Hamadan” accession measured. produced 2128 g/m2 of fresh yield compared to four Quantitative characteristics studied in this study native populations of “Shirvan”, “Karaj”, “Varamin”, included germination percentage (when 50 percent of and “Borojerd” accessions (Ahmadi et al., 2010). the seeds was germinated), leaf length (cm), leaf width However, neither study has been done to identify (cm), petiole length (cm), petiole diameter (mm), leaf Iranian accessions in spring planting, nor have area (mm2), plant height (cm), fresh and dry weight (g). morphological comparisons of these masses. In order to calculate the yield of the accessions, after Considering spinach bolting in spring cultivation, it is randomly harvesting, plants were weighed and their important to study it in Isfahan climate conditions. This fresh weight was calculated in grams per plant and study was conducted based on the amount of spinach finally their average performance which was considered cultivation in Iran and the need for farmers to produce as yield (kg ha-1). In order to estimate the economic high-yielding populations. This study also compared yield, the equivalent level of each replication was commercial populations of Iranian and foreign spinach considered. After harvesting and separating the leaves, accessions. One of the main objectives of this study was to they were weighed and their economic yield was evaluate the most desirable traits of spinach, such as yield, calculated in kg per hectare, and leaf numbers, male and economic benefits, late bolting (a desirable feature in female plants percentage, days to flowering were spinach) and male plants traits of Iranian populations evaluated. In order to measure the shoot dry weight, it compared to foreign populations. The authors believe that was placed in an oven at 70 °C for 48 hours. this study can lead to determining the best suitable Also, the qualitative features were also studied based accession for spring cultivation in the conditions of the city on descriptors by Bioversity International Plant Genetic of Isfahan as well as finding the best accession for the Resources Institute (IPGRI) presented in Table 2 (Arif future breeding program. et al., 2013; Jafari and Jalali 2017). These traits consist

of type and colour of seed, stem anthocyanin, petiole and leaf attitude, wrinkles of leaf, leaf thickness, leaf MATERIALS AND METHODS sheath attitude, leaf shape, leaf colour, lobed leaf tip, the

shape of leaf tip and wave margin of leaf. Plant Material

At first in this study, 26 Iranian spinach accessions were Analysis Data collected from the seed and plant improvement institute Data were tested by one-way analysis of variance and the gene bank of Iran (SPII). It should be noted that (ANOVA) and analyzed using SAS software (version 9.4). endemic accessions in this research were selected based Significant differences between means were tested on the results of a study (Jafari and Jalali, 2017) considering the least significant difference (LSD) test at P conducted on 107 Iranian spinach accessions. They < 0.05 level of probability. The correlation of the traits was reported that masses of “Varamin88”, “Hamedan”, also tested using SAS 9.4 software. Clustering of 44 “Lorestan”, “Varamin Prickly”, “Varamin Advanced spinach accessions was done using Statgraphics Centurion Prickly” and some other accessions were favorable on (XVII) software. The biplot diagram of the accessions was the basis of agronomic and morphological features, and investigated with the help of the R-software. these accessions were used in the present study to accurately evaluate the yield, bolting and compare them with foreign trade accessions. The 18 foreign accessions RESULTS AND DISCUSSION were also provided by the Leibniz Institute of Plant

Genetics and Crop Plant Research (IPK) gene bank Qualitative Traits (IPK, 2018). Details of these accessions are shown in Table 1. Seeds of the accessions, in an experiment based Distribution of qualitative traits in this study based on on a randomized complete block design (RCBD) with spinach descriptor is presented in Table 2 (Arif et al., three replications, were sown in a field located at 2013; Jafari and 2017). Among the accessions of this Isfahan University of Technology, Isfahan, Iran, in study, “Alwaro” samples were not germinated in any of March 2018 (spring sowing). Because soil organic the replications and so, were completely eliminated. 88

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Table 1. The characteristics of 44 spinach accessions which were collected from different regions in the world and used in this study Accession Collecting Altitude Accession Collecting Longitud Altitude Accession Collecting Altitude No. Longitude Latitude No. Latitude No. Longitude Latitude name place (meter) name place e (meter) name place (meter) 1 TN-69-22 Yazd 54◌ 21' E 31◌ 53' N 1215 16 TN-69-138 Lorestan5 48◌ 55' E 33◌ 45' N 1347 31 Subito Netherland _ _ _ 2 TN-69-31 Fars 52◌ 22' E 29◌ 37' N 1540 17 TN-69-139 Lorestan6 48◌ 29' E 33◌ 43' N 1148 32 Alwaro Germany _ _ _ Virginia Savoy 3 TN-69-57 Hamedan1 48◌ 31' E 34◌ 48' N 1850 18 TN-69-140 Lorestan7 48◌ 24' E 33◌ 45' N 1179 33 USA _ _ _ Blight Resistant Virginia Savoy 4 TN-69-58 Lorestan1 48◌ 21' E 33◌ 29' N 1200 19 TN-69-149 Hamedan4 48◌ 51' E 34◌ 82' N 1774 34 USA _ _ _ Fusarium Chaharmahal 5 TN-69-70 Hamedan2 48◌ 51' E 34◌ 79' N 1810 20 TN-69-153 50◌ 86' E 32◌ 32' E 2061 35 Garant Germany _ _ _ and Bakhtiari Razavi 6 TN-69-73 Kerman 57◌ 05' E 30◌ 17' N 1775 21 TN-69-163 59◌ 36' E 36◌ 18' N 979 36 Viking Italy _ - _ Khorasan 7 TN-69-74 Lorestan2 48◌ 35' E 33◌ 48' N 1118 22 TN-69-172 Qazvin 50◌ 72' E 36◌ 27' N 1310 37 Viroflay Germany _ _ _ Varamin 8 TN-69-76 Hamedan3 48◌ 52' E 34◌ 77' N 1833 23 Tehran 51◌ 28' E 35◌ 70' N 1173 38 Spinagh Iraq _ _ _ Prickly SPI 108(IPK 9 TN-69-78 Tehran 51◌ 38' E 35◌ 23' N 1214 24 Varamin 88 Tehran 51◌ 39' E 35◌ 19' N 915 39 Afghanistan _ _ _ No.) Sistan and SPI 211(IPK 10 TN-69-79 60◌ 85' E 29◌ 50' N 1386 25 Kashan Kashan 51◌ 27' E 33◌ 59' N 950 40 Turkey _ _ _ Baluchestan No.) Varamin SPI 79(IPK 11 TN-69-81 Qom 50◌ 53' E 34◌ 38' N 930 26 Advanced isfahan 52◌ 02' E 32◌32' N 1525 41 Azerbaijan _ _ _ No.) Prickly East 12 TN-69-88 46◌ 18' E 38◌ 04' N 1366 27 Matador Germany _ _ _ 42 Late Flower China _ _ _ Azerbaijan1 East 13 TN-69-97 46◌ 30' E 38◌ 07' N 1402 28 D’inverno Italy _ _ _ 43 Tenjinmaru Japan _ _ _ Azerbaijan2 14 TN-69-101 Lorestan3 48◌ 36' E 33◌ 47' N 1274 29 Monatol Germany _ _ _ 44 Kasi Jong South Korea _ _ _ 15 TN-69-137 Lorestan4 48◌ 33' E 33◌ 46' N 1164 30 Wiremona Germany _ _ _ *All foreign accessions were provided from IPK gene bank (IPK, 2018)

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Results of the study of qualitative traits divided the three characteristics of leaf standing, petiole standing seeds into two groups of smooth and prickly of which, and no shrinkage or low shrinkage, which can be used 32.5% of the total seeds had prickly seeds, and the rest in breeding programs suitable for mechanical harvesting had smooth seeds (Table 2). The results of studied traits (Table 3). The “Spinagh” also possessed these three for both smooth and prickly seeds were similar to the characteristics together, but it had a triangular leaf shape results found by previous researches (Asadi and (intense arrow) that was not desirable. Hasandokht, 2007; Eftekhari et al., 2010). Dominant In general, it can be said for mechanical harvesting, seed colors were gray-green and gray. Commercial Iranian accessions were superior to the foreign ones due accessions of spinach have a smooth seed. In spite of to better petiole and leaf form, and low shrinkage of that, prickly seed during mechanical planting have an leaves. It has been reported in foreign countries that uneven distribution (Asadi and Hasandokht, 2007). wrinkled leaves of spinach are more marketable than flat leaves (Kuwahara et al., 2014). In confirmation of Table 2. Distribution of qualitative traits in this study based this report, the majority of foreign populations of Table on spinach descriptor 3 have wrinkled leaves. Usually, spinach cultivars with Plant trait Description Frequency (%) lower leaf shrinkage have less nitrate content and there Smooth 29 67.5 is a direct correlation between leaf shrinkage and nitrate Seed type Prickly 14 32.5 content (Arshi, 2000). So no-shrinkage leaves as a Yellow-green 11 25.5 desirable attribute is of interest to researchers. While in Seed color Gray-green 15 34.8 Iran, the most favorable spinach has a broad, fleshy, Gray 17 39.5 thick and juicy leaf, and according to Table 3, the Stem Yes 1 2.3 endemic accessions of “Varamin Prickly” and Anthocyanin No 42 97.6 “Varamin88” have these characteristics. In Iran, the Erect 9 20.9 Petiole Semi-erect 22 51.1 optimal form of spinach leaf is round and in overseas attitude Horizontal 12 27.9 triangular shapes has been reported to be desirable High 6 13.9 (Kunicki et al., 2010). Wrinkles of Intermediate 10 23.2 Leaf color is very important in leafy vegetables leaf Low 17 39.5 since the presence of green pigments is a desirable and

Smooth 10 23.2 marketable feature for frozen spinach (Eftekhari et al., Very thick 3 6.9 2010). Researchers have reported that in spinach, the Leaf Thick 7 16.2 green color is usually associated with chlorophyll a and thickness Intermediate 29 67.4 b pigment which prevent the appearance of carotenoid Fragile 4 9.3 pigments (Kidmose et al., 2005). According to data Erect 17 39.5 Leaf Sheath Semi-erect 14 32.5 presented in Table 3, leaf color was variable from very attitude Horizontal 12 27.9 dark green to light green, and ideal foreign accessions Oval 15 34.8 had darker leaves than the Iranian ones. In Iranian Leaf shape Broad oval 16 37.2 accession, “Hamedan2”, “Varamin Prickly” and Triangular 12 27.9 “Varamin88” had some dark green color (Table 3). In Very dark most of the studied accessions in this study, the edge of green 7 16.2 the leaf was flat, the tip of the leaf was round, and 51.1% Leaf color Dark green 4 9.3 of the accessions had a wave at the margin of their Intermediate 19 44.1 leaves (Tables 2 and 3). green 13 30.2

Light green Concave 14 32.5 Quantitative Traits Leaf Sheath Bulgy 6 13.9 In this study, it was shown that high agronomic and Flat 23 53.4 morphological diversity exists for most of the measured Bend 9 20.9 Lobation of traits that may be useful to future researchers. The range Flat 25 58.1 leaf tip Upward 9 20.9 of variance among the accessions examined in this study The shape of Sharp 9 20.9 indicated the presence of genetic diversity among leaf tip Angled 15 34.8 Iranian and foreign accessions. Analysis of variance of Circular 19 44.1 quantitative traits showed that the accessions in the Wave margin Yes 22 51.1 aspect of all morphological characters were different at of leaf No 21 48.8 the 1% level (Table 4). In this study, Iranian accessions (Yazd, East Results of other qualitative traits are presented in Azerbayjan1, Lorestan 4, 5 and 6, Varamin88, Varamin Table 3. It has been reported that accessions that have a Prickly, Varamin Advanced Prickly) were better than petiole standing, wrinkled or slightly wrinkled, were foreign accessions concerning leaf characteristics such suitable for mechanical harvesting (Splittstoesser, 1990). as leaf number, leaf length and width, petiole length and Accordingly, accessions of “Matador”, “D’inverno”, diameter, leaf area and plant height (Table 5). Leaf area and “Monatol” with good performance are unsuitable showed a positive correlation with germination for mechanized harvesting because their petiole and leaf percentage (Table 6). It was stated that if spinach has a forms are compact. Accessions of “Kashan”, “East better germination, it would result in greater leaf area Azarbaijan1”, “Lorestan4” and “Lorestan7” have all and ultimately more yield (Turner, 2001). Zaferaniye 90

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(2015) observed that the most desirable characteristics “Varamin” population. of spinach leaves, such as leaf number, were in the

Table 3. Qualitative properties of Iranian and foreign spinach accessions stem Leaf Lobati The Wave Accession Seed Seed Petiole Wrinkles Leaf Leaf Leaf Leaf Anthocya- Sheath on of shape of margin name type colour attitude of leaf thickness shape colour Sheath nin attitude leaf tip leaf tip of leaf TN-69-22 P YG N SE L I SE TR IG F F SH N TN-69-31 P YG N SE L I SE TR IG C UW SH N TN-69-57 S YG N H I I H TR IG C UW A Y TN-69-58 S GG N H L I H BO LG F F CI N TN-69-70 S YG N H L I H TR DG C UW CI Y TN-69-73 S GG N SE L I E BO LG F F A N TN-69-74 S YG N SE L I E O IG F F CI Y TN-69-76 S GG N SE L I SE TR IG B F A Y TN-69-78 P GG N SE L I H O LG F F CI N TN-69-79 S GG N SE L I SE O LG F F CI N TN-69-81 S GG N SE L I E BO LG F F SH N TN-69-88 S GG N E S I E BO LG F F SH Y TN-69-97 S GG N SE S I E TR IG F F SH Y TN-69-101 S GG N SE L I SE O IG F F A Y TN-69-137 S GG N E L I E BO IG C B CI N TN-69-138 S GG N E L FR E BO IG B B A Y TN-69-139 S GG N SE I I SE BO IG C B CI Y TN-69-140 S GG N E S I E BO IG F F A N TN-69-149 P YG N SE S I SE TR LG F F SH N TN-69-153 S YG N SE L T SE O LG F F A N TN-69-163 S YG N SE S I E O LG F F A N TN-69-172 P YG Y SE S I E O LG F F A Y Varamin P GG N E I T E BO DG C B CI Y Prickly Varamin S GG N E I T E BO VDG B B CI Y 88 Kashan S YG N E L FR E TR IG C F A N Varamin Advanced P YG N SE L T E TR IG C B CI N Prickly Matador S G N H H VT H O IG C UW CI N D’inverno S G N H H VT SE BO VDG C UW CI Y Monatol S G N H H VT SE BO VDG C UW CI N Wiremona S G N H H T H O VDG C UW CI Y Subito P G N H I T SE O IG B UW CI Y Virginia Savoy S G N H H I H BO VDG B B CI N Blight Resistant Virginia Savoy S G N H H I H BO VDG B B CI N Fusarium Garant S G N H I I H O LG F F CI N Viking S G N H I I H O LG F F A Y Spinagh P G N E L FR E TR LG F F SH Y Viroflay S G N SE I T SE BO VDG C UW CI Y SPI 108(IPK P G N SE I I SE O DG F F A N No.) SPI 211(IPK P G N SE S I SE O IG F F SH Y No.) SPI 79(IPK S G N SE I I H BO DG C B A Y No.) Late P G N SE S I H TR IG F F A Y Flower Tenjinmar P G N SE S I E TR IG F F SH Y u Kasi Jong P G N E S FR E O IG F F A N Abbreviation signs: Smooth (S), Prickly (P), Yellow-green (YG), Gray-green (GG), Gray (G), Yes (Y), No (N), Erect (E), Semi- erect (SE), Horizontal (H), High (H), Intermediate (I), Low (L), Very thick (VT), Thick (T), Fragile (F), Oval (O), Broad oval (BO), Triangular (TR), Very dark green (VDG), Dark green (DG), Intermediate green (IG), Light green (LG), Concave (C), Bulgy (B), Flat (F), Bend (B), Upward (UW), Sharp (SH), Angled (A), Circular (CI).

Abolghasemi et al. / Iran Agricultural Research (2019) 38(1) 87-99

Table 4. Analysis of variance of traits in different accessions of spinach Source MS of Percent Leaf Petiole Germinati Percent Variati df Plant Leaf Leaf Petiole Fresh Dry Days to of num diamete Yield Leaf area on of Male on height length width length weight weight flowering Female ber r percentage plant plant 135.2 Blocks 2 7.54 4.53 1.72 2.76 0.93 618266 60.49 0.41 84587 71.79 65.60 5.32 19.60 6 Access 42 31.75** 144.28** 21.57** 5.98** 25.75** 3.15** 160043** 2337.41** 59.21** 11722998**1265.47** 289.98** 525.77** 535.58** ions Error 84 6.85 12.32 2.10 0.87 1.59 0.43 33399 126.26 3.76 496245 9.51 1.56 5.26 6.15 Total 128 14.0 CV 19.66 18.37 12.38 12.90 18.39 26.11 26.11 24.79 11.15 14.90 14.97 16.10 13.97 5 ** Statistically significant at one-percent level of significance.

The petiole length showed a high correlation with plant (0.56**) (Table 6). These results of correlations accord height (0.85**) and the leaf length (0.70**) (Table 6). The with the result of Varalakshmi and Devaraju (2010) leaf length, leaf width and number of leaves are important about the positive relationship in morphological traits. parts of yield in spinach (Asadi and Hasandokht, 2007). As According to Zaferaniye (2015), there is a positive it is observed in this study, accessions such as “D’inverno”, relationship between yields, fresh and dry weight, leaf “Monatol”, “Viroflay”, “Matador”, “Varamin88”, area and number of leaves of spinach. “Varamin Prickly” and “Lorestan5” with a high number of In the present study, most of the accessions which have leaves had more yield (Table 5). Accessions with more good dry weight had a low shrinkage in leaf and only the leaves that enter the reproductive stage usually have more “Spinagh”, “Kashan” and “Varamin Advanced Prickly” fresh and dry weight and better performance and can be accessions had a flat leaf (Tables 3 and 5). One of the used with late bolting goals in breeding actions (Asadi and desirable traits for spinach processing and packaging is dry Hasandokht, 2007). As the correlation table of the traits weight and it has been reported that the high dry matter has shows, the number of leaves has a completely positive a direct relationship with the flat leaf in spinach (Arshi, relationship with yield and days to flowering (Table 6). By 2000; Eftekhari et al., 2010). In this study, the dry weight Jafari and Jalali (2017) are also approved a positive of the accessions was very different (Table 5). It can be correlation between leaf number, yield and fresh and dry argued that the difference in water content in the tissues of weight of spinach. different accessions leads to a difference in dry weight. A The highest percentage of yield was observed in the few researchers have reported that the highest dry weight foreign accession of the “Viroflay” (71.224 ton/ha) and was observed in “Qom” accession, which differed from the the “D’inverno” (64.259 ton/ha), respectively; however, results of the current study. It could be due to differences in no significant differences were observed between these the growing season (Asadi and Hasandokht, 2007). accessions (Table 5). Then, the native accession of Because in the autumn crop, despite the long growing “Varamin88” with 52.590 ton/h was better than other season, cold and moisture stress affected the dry weight of Iranian accessions. The fresh and dry weights of the the spinach. plants were not significantly different between The germination percentage of “Monatol”, “Kashan”, “Varamin88”, “Matador” and “D’inverno” accessions In and “D’inverno” accessions were better than the others Isfahan climate conditions, “Varamin88” is (Table 5). Contrary to our results, in another study, recommended to the farmers for cultivation (Table 5). “Varamin” had the highest percentage of germination In addition, “Varamin Prickly” and “Varamin Advanced (Zaferaniye, 2015). This could be due to the absence of Prickly” have a high potential yield and can be used to “Kashan” accession in their study. improve the performance of spinach since these Spinach is a leafy vegetable; so, a long time for accessions were considered valuable concerning growing it is favorable. Researchers have divided the breeding and gardening programs (Eftekhari et al., 2010; spring spinach flowering into three groups including Morelock et al., 2008; Stagnari et al., 2007). Similar less than 60 days flowering (early flowering), 60 to 70 results have been reported on the benefit of the days flowering (medium flowering) and more than 70 performance of “Varamin88” and “Varamin Prickly” days flowering (long flowering) (Chitwood et al., 2016). accessions (Asadi and Hasandokht, 2007; Eftekhari et The highest growth period among the available al., 2010). Researchers in a study in New Zealand found accessions was 89.60 days that belonged to the” that the harvest of spring planting was less than the fall D’inverno” accession and then the “Viroflay”, planting. They stated that one of the main reasons for “Matador”, “Hamedan2” and “Spinagh”, respectively, reducing the yield of spring crops is to increase the were among the long-flowering groups in the weather of length of the day and to enter the reproductive stage Isfahan (Table 5). The medium flowering groups in this (Chunilall et al., 2006). According to the correlation study included “Varamin88”, “Varamin Prickly”, trait, there is a positive correlation between yield and “Varamin fresh weight (0.98**), dry weight (0.82**), leaf number (0.66**), leaf area (0.63**) and day to flowering 92

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Table 5. Comparison of the average variance of leaf characteristics of native and foreign spinach accessions Leaf Leaf length Leaf width Petiole Petiole Yield Fresh Dry Leaf area Germination Days to Male Plant height Female Accession No. number (cm) (cm) length (cm) diameter (mm) (kg/h) weight(g) weight (g) (mm2) percentage (%) flowering plant (%) (cm) plant (%)

Yazd 12.13h-n† 16.53h-r 12.80e-j 9.20a-e 9.07e-k 4.82a-d 21079f-j 40.98f-j 8.77e-j 8363bc 45p 66.33h-j 37.70n-q 61.96g-j Fars 10.33l-n 15.46i-r 14.13c-f 7.53f-k 9.26e-i 4.31c-g 12879j-n 25.04j-n 3.43op 7601b-f 56.85lm 61.66no 37.82m-q 61.51g-k Hamedan1 17.13b-f 14.66k-r 12.73e-j 7.20g-l 7.60g-q 4.27c-g 15347j-n 29.85j-n 8.87e-i 6751e-j 61.11j-l 68.00f-h 31.65rs 66.45f Lorestan1 12.93f-n 10.86r 11.60g-n 6.73h-o 6.60m-r 3.78d-m 13688j-n 26.61j-n 4.15n-p 6175g-m 71.88h 67.66f-i 44.09f-j 56.21m-q Hamedan2 15.86c-h 16.60h-q 12.60f-j 7.80e-j 7.20j-q 3.87c-k 15798j-n 30.72j-n 6.55h-o 6806e-i 61.44i-l 77.00d 45.95e-h 53.71o-s Kerman 9.60mn 13.53m-r 9.46n-r 7.13g-m 8.26f-n 2.54n-r 13391j-n 26.03j-n 2.71p 7254c-g 66.32i 63.00l-o 45.04e-i 55.38m-r Lorestan2 10.93j-n 19.86f-l 11.20h-o 6.13k-p 10.06d-f 2.37o-r 16877i-n 32.81i-n 4.29n-p 5981i-n 77.10fg 61.00o 48.26c-e 52.28q-t Hamedan3 10.40l-n 21.06f-i 11.40g-o 8.00d-i 8.93e-l 3.49f-n 17357h-n 33.74h-n 5.17 l-p 7139d-h 61.77i-l 63.00l-o 34.52qr 65.21fg Tehran 10.66j-n 15.33j-r 9.53n-r 6.20k-p 7.40i-q 3.39g-o 14100j-n 27.41j-n 5.30 l-p 4353o-s 38.54q 65.00j-l 48.05de 50.69s-v Sistan 11.20i-n 11.86n-r 8.40qr 6.66i-o 7.73g-q 2.74m-r 11228k-n 21.83j-n 4.29n-p 3973q-t 44.10p 65.00j-l 38.10m-q 61.56g-k Qom 10.53k-n 14.80k-r 8.06qr 6.53i-o 9.06e-k 2.78l-r 12029J-n 23.39j-n 4.98 l-p 4872n-r 63.44i-k 49.00r 39.72k-o 57.60k-o East.Az.1 10.40l-n 19.93f-l 10.20k-q 6.73h-o 9.20e-j 3.06h-p 11380k-n 22.13k-n 3.63op 8422b 92.80bc 52.33q 46.62e-g 53.07p-t East.Az.2 10.80j-n 17.66f-l 12.06f-l 7.00g-n 8.40e-m 4.12c-h 15642j-n 30.41j-n 5.67j-p 6014h-n 51.00qr 51.00qr 43.86f-j 53.18p-t Lorestan3 10.40l-n 17.60h-m 13.26e-i 8.20c-h 10.33de 4.54b-f 13300j-n 25.86j-n 3.71op 7242c-g 54.88m-o 57.00p 46.68e-g 53.33p-t Lorestan4 12.26h-n 20.33f-k 13.66d-g 8.33b-g 13.06bc 3.89c-j 27795e-g 54.03e-g 5.12 l-p 7395b-f 65.66ij 56.66p 48.58c-e 51.58r-u Lorestan5 19.06a-d 28.33cd 17.53a 9.80ab 12.40c 4.91a-c 26473e-h 51.47e-h 5.77i-p 7613b-f 91.10c 49.00r 30.10s 70.77e Lorestan6 17.20d-i 31.26bc 17.00ab 9.53a-c 15.06ab 3.90c-j 25429e-i 49.44e-i 5.30l-p 6514f-k 72.88gh 51.33q 60.30a 39.06w Lorestan7 12.00h-n 27.46c-e 13.33e-h 7.13g-m 11.46cd 5.56ab 17146h-n 33.34h-n 3.31p 7118d-i 80.66ef 51.33q 51.52b-d 48.23uv Hamedan4 12.93f-n 17.66h-m 11.26h-o 7.33g-l 7.46i-q 3.73e-m 18162h-n 35.31h-n 7.93g-l 5508k-n 72.89h 55.33p 51.45cd 50.85s-v Chaharmahal 12.40g-n 15.46i-r 12.26f-k 7.80e-j 8.00g-o 4.06c-h 12572j-n 24.44j-n 4.50m-p 6240g-l 44.77p 55.00p 42.51h-l 56.89l-p Khorasan 12.00h-n 17.33h-o 10.93i-p 6.53i-o 7.60g-q 2.80k-q 13129j-n 25.53j-n 5.61k-p 5054m-q 65.66ij 55.66p 35.95pq 63.71f-h Qazvin 10.06mn 17.00h-p 10.86j-p 5.40op 8.40e-m 3.08h-p 15278j-n 29.71j-n 3.66op 6038h-m 38.88q 56.00p 42.99g-k 58.19j-n Var-Prickly 15.66c-h 25.00d-f 13.73d-g 7.33g-l 12.66c 4.35c-g 30867e 60.02e 8.63f-k 7120d-i 64.71i-k 69.66f 51.86bc 49.59t-v Var88 15.80c-h 29.40b-d 16.40a-c 8.86b-f 12.93c 3.81d-l 52590b 102.25b 15.84b 10658a 83.88de 72.00e 38.10m-q 60.46h-l Kashan 14.40e-l 45.06a 15.86a-d 9.13a-e 15.73a 4.00c-i 34204de 66.50de 9.62d-h 7966b-d 97.56ab 43.00s 47.11ef 56.97l-p Var.Adv.Prickly 10.00mn 35.06b 15.06b-e 9.40a-d 16.20a 4.64b-e 28809ef 56.02ef 9.20d-h 7121d-i 91.09c 61.33o 35.67pq 62.96f-i Matador 19.66a-c 20.60f-j 15.00b-e 10.60a 7.06k-q 5.81a 48436bc 94.18bc 16.11b 8490b 66.99ij 83.66c 36.06o-q 62.86f-i D’inveno 22.53a 21.86e-h 15.86a-d 9.06b-e 9.60d-g 5.78a 64259a 124.95a 15.79b 11483a 96.03a-c 89.66a 23.63t 76.39d Monatol 20.20ab 19.66f-l 12.00f-m 9.06b-e 6.20o-s 4.75a-e 42056cd 81.77cd 14.77bc 10781a 98.58a 69.00fg 3.82w 95.96a Wiremona 12.80g-n 17.46h-n 11.20h-o 7.40f-l 4.33s 3.96c-j 14854j-n 28.88j-n 5.40l-p 6185g-m 63.2i-k 67.33g-i 7.00vw 94.42ab Subito 10.40l-n 13.73m-r 8.33qr 5.93l-p 5.86q-s 2.96i-q 10050mn 19.54mn 3.33p 4300p-s 63.34i-k 64.00k-m 14.43u 88.29c Virginia Savoy 12.20h-n 11.13qr 7.30r 5.53n-p 4.93rs 2.93j-q 8870n 17.24n 8.99e-h 2884st 20.24r 62.00m-o 40.58j-n 59.23i-m blight resistant Virginia Savoy 12.06h-n 13.06m-r 9.20o-r 5.66m-p 7.93g-p 2.40o-r 10667l-n 20.74j-n 5.54k-p 3393st 60.33kl 61.33o 39.17l-p 62.16g-j fusarium Garant 12.53g-n 14.26l-r 9.06o-r 6.53i-o 6.00o-s 2.21p-r 12923j-n 25.12j-n 7.47h-m 4196p-s 50.05o 63.00l-o 6.65vw 93.66ab Viking 15.73c-h 11.40p-r 8.73p-r 5.66m-p 5.86q-s 2.77l-r 11632k-n 22.61k-n 10.94d-g 3452st 22.89r 63.66k-n 9.80v 90.77bc Viroflay 16.60b-c 18.73g-m 13.47e-h 7.40f-l 9.53d-h 3.81d-l 71224a 138.49a 23.79a 7801b-e 85.86d 87.33b 29.77s 71.47e Spinagh 18.46a-e 24.33d-g 12.00f-m 8.00d-i 12.93c 2.90j-q 34503de 67.08de 12.21cd 5660j-n 91.78c 76.00d 51.18b 47.49v 93 Abolghasemi et al. / Iran Agricultural Research (2019) 38(1) 87-99

Table 5. Leaf Leaf length Leaf Petiole Petiole Yield Fresh Dry Leaf area Germination Days to Male Plant height Female Accession No. number (cm) width length (cm) diameter (kg/h) weight(g) weight (g) (mm2) percentage (%) flowering plant (%) (cm) plant (%) (cm) (mm) SPI108 8.93n 14.60l-r 8.40qr 6.46j-o 5.93p-s 2.56n-r 20531f-k 39.92f-k 11.51d-f 5484k-o 61.98i-k 66.66h-j 28.23s 72.45de SPI211 9.53mn 16.73h-q 9.73l-q 5.93l-p 6.33n-s 2.30P-R 25393e-i 49.37e-i 10.98d-g 5135l-p 54.54m-o 62.33m-o 41.53i-m 59.20i-m SPI79 14.66e-k 16.93h-p 9.33n-r 5.46op 6.73m-r 3.38g-o 19908f-l 38.71f-l 11.89c-e 5199l-p 51.54no 69.00fg 34.85qr 63.84f-h Late flower 12.86g-n 11.73o-r 7.26r 5.53n-p 6.93l-r 2.28p-r 14467j-n 28.13j-n 7.17h-n 3750r-t 18.40r 65.66i-k 46.90ef 54.67n-s g- e-j l-r m-q p h-q qr 18968 g-m h-o p-s q h-j s e Tenjinmaru 14.80 14.33 9.66 4.93 7.53 1.90 m 36.88 6.56 4134 38.90 66.66 28.78 71.30 Kasijong 13.66f-m 24.20d-g 11.66g-n 5.26op 12.86c 1.74r 16431i-n 31.95i-n 8.05g-l 4117p-s 55.16mn 67.33g-i 46.94ef 51.86r-u LSD5% 4.25 5.69 2.35 1.52 2.05 1.06 9383 18.24 3.14 1266.2 5.00 2.03 3.72 4.02 † Different letters defined in each column mean significant differences at the 5% level.

Table 6. The correlation coefficient of quantities traits investigated in 43 spinach accessions Traits Leaf Plant Leaf Leaf Petiole Petiole Yield Fresh Dry Leaf area Germination Day to male female number height length width length diameter weight weight percentage flowering plant plants Leaf number 1 Plant height 0.28ns 1 Leaf length 0.51* 0.74** 1 Leaf width 0.48** 0.60** 0.82** 1 Petiole length 0.16ns 0.85** 0.70** 0.5ns 1 Petiole 0.45* 0.40* 0.73** 0.78** 0.27ns 1 diameter Yield 0.66** 0.45* 0.56** 0.5** 0.35* 0.42* 1 Fresh weight 0.66** 0.45* 0.59** 0.55** 0.34* 0.47* 0.98** 1 Dry weight 0.61** 0.19ns 0.25ns 0.28ns 0.03ns 0.23ns 0.82** 0.84** 1 Leaf area 0.45* 0.48** 0.73** 0.76** 0.37* 0.73** 0.63** 0.67** 0.37* 1 Germination 0.38* 0.65** 0.62** 0.60** 0.53** 0.44* 0.58** 0.57** 0.28ns 0.69** 1 percentage Day to 0.50** -0.22ns 0.04ns 0.08ns -0.24 0.14ns 0.56** 0.58** 0.67** 0.24ns 0.05ns 1 flowering male plants -0.21ns 0.26ns 0.19ns 0.03ns 0.5** -0.005ns -0.11ns -0.11ns -0.29ns -0.04ns 0.02ns -0.29ns 1 Female plant 0.2ns -0.25ns -0.2ns -0.04ns -0.5ns -0.006ns 0.11ns 0.11ns 0.28ns 0.03ns -0.01ns 0.28ns -0.99ns 1 *: Significant at 5% probability levels. **: Significant at 1% probability levels

94 Abolghasemi et al. / Iran Agricultural Research (2019) 38(1) 87-99

Advanced Prickly” and foreign accessions consisted of accessions “Varamin Prickly and Varamin Advanced “Monatol”, “Wiremona” and “Late flower”. In the Prickly, Kashan, Lorestan 4, 5, 6, 7, Spinagh”, and present study, 50% of Iranian accessions were placed in “Viking, Garant, Subito, and Wiremona” and other the group of early flowering, the earliest of which was accessions were placed in separate subgroups. In spite “Kashan” with a growth period of 45 days. On the other of the morphological difference between 43 accessions hand, none of the foreign accessions was early in this study, accessions with similar characteristics flowering (Table 5). Similar to our results, in the study belonging to different regions of the world could be of Zaferaniye (2015), the Iranian masses of spinach grouped together. In subgroups of cluster П, accessions were the fastest flowering masses and foreign masses of “Wiremona”, “Subito”, “Garant” and “Viking” were were the latest flowering. Researchers have reported placed together, with the most similarity of these that late flowering spinach is mostly favorable for accessions being the high percentage of female plant economic production because their flower stems appear (Fig 1). “Kashan” accession in cluster П, although had slowly (Asadi and Hasandokht, 2007; Kawazu et al., some desirable traits, goes to flower soon. The best 2003). Different results have been reported for day to accession from cluster П was “Varamin Advanced flowering, which may be due to the seasonal mismatch Prickly” (endemic accession) which had favorable between the studies conducted (Asadi and Hasandokht, morphological traits. Other accessions in cluster П 2007). The flowering of spinach occurs early in spring subgroups a little differed in qualitative and quantitative planting due to weather conditions, long day and high traits so that their features were not desirable. temperatures, which is not seen in autumn cropping Geographically, accessions collected from different (Asadi and Hasandokht 2007). Because the spinach parts of Lorestan and Hamedan provinces were placed plant produces both male and female flowers, spinach almost in the same subgroups. For the effective use of spring planting is important if the plant can have female spinach in any breeding or cultivating program, an flowers (Zaferaniye, 2015). accurate assessment of various indigenous populations Contrary to the reports of other researchers (Arshi, is essential. 2000; Asadi and Hasandokht, 2007; Morelock et al., To better determine the best accessions and to 2008), in this study, spinach was not observed in investigate the relationships between all agro- dioecious plants and all plants were pure males or pure morphological features, there is a need to biplot the female. It seems that the monoecious plant is likely spinach traits. So with the help of (R) software, 43 found in the ancestors or wildlife of this plant. Spinach accessions were checked by biplot classification whose female plants are valuable in planting; so, these plants results fully accorded with clustering analysis (Fig 2). are used in breeding programs and seed production PC analysis used in this research has been useful in (Stagnari et al., 2007). grouping germplasm in many herbs such as blackpea (Ghafoor et al., 2001), chickpea (Naghavi and Relationship Among Genotypes by Cluster analysis Jahansouz 2005), rice (Tripathi et al. 2013) and and Biplot sorghum (Barro-Kondombo et al., 2010). Based on biplot, accessions located in region (D), Identification and selection of indigenous spinach had inappropriate (negative) characteristics which were populations and their comparison with foreign not considered in the range of useful traits. Most of the populations is one of the most important methods for Iranian accessions were located in this area (D). As it is evaluating, screening and preserving the genetic observed, these accessions were inappropriate resources of this plant (Ebadi-Segheloo et al., 2014). In concerning both vegetative yield and mechanical traits. addition to the mean comparison of traits, the accessions The accessions located in region (C) were negative for were also divided according to cluster analysis (Fig 1). the first component (dimension 1) but were positive for Cluster analysis can be useful for selecting high-quality the second component (dimension 2). These accessions accessions for agronomic and breeding purposes. The consisted of the most foreign spinach such as “Garant”, favorable accession selection in cluster analysis is based “Viking”, “Wiremona” and etc. These accessions in on the greater distance between the accessions because region (C) were the rosette form with the high the short distance between them represented the slight percentage of the female plant. Indeed, accessions in difference. region (C) were very poor for mechanical harvesting. The cluster analysis divided the studied accessions The accessions located in region (B) were positive for into two major groups (cluster I and П) at a distance of the first component (dimension 1) and negative for the 35 to 60. “Monatol”, “Viroflay”, “D’inverno”, “Matador” second component (dimension 2), which had favorable and “Varamin88” accessions were grouped in one leaf appearance characteristics and suitable for cluster (Fig 1). All of these accessions (cluster I) had a mechanical harvesting so that these accessions had smooth seed, no anthocyanin in the stem and had a common traits of plant height, leaf and petiole length, desirable performance compared to others. In terms of and leaf width. In this region (B), the most suitable flowering time, they were placed in the late and medium accessions for mechanical harvesting were “Kashan”, flowering group. In general, the accessions in cluster I “Lorestan5 and 6”, Varamin Advanced Prickly” and were superior to cluster П in morphological traits. Other “Varamin Prickly”. On the other hand, due to the accessions of this study were placed in a large cluster i.e. presence of the significant male plant in these cluster П. Cluster I and П had a little genetic similarity, accessions, the second component (dimension 2) was and the accessions placed in cluster П also had different placed in negative conditions. The accessions located in variations of features. As can be seen in Fig. 1, region (A) were positive for the first and second 95

Abolghasemi et al. / Iran Agricultural Research (2019) 38(1) 87-99 components (dimension1 and 2). These accessions which is consistent with the results of clustering (located in region A) had favorable economic analysis. It is worth noting that the scattering pattern of characteristics and a high percentage of the female plant. the accessions is shown in Fig. 3. This pattern Finally, it can be said that the optimal accessions such completes previous evaluations and this result has also as “D’inverno”, “Viroflay”, “Monatol”, “Matador” and approved the bi-plots diagram. “Varamin88” have the best conditions in this study,

Fig. 1. Cluster analysis of the 43 spinach accessions based on Average method using measured traits.

Fig. 2. Biplot diagram of 43 endemic and external spinach accessions. Most of the Iranian accessions were located in this area (D); which were not considered in the range of useful traits. Accessions in region (C) were the rosette form with the high percentage of the female plant. Accessions in region (B) had common traits of plant height, leaf and petiole length, and leaf width. Accessions located in region A had favorable economic characteristics and a high percentage of the female plant.

Abolghasemi et al. / Iran Agricultural Research (2019) 38(1) 87-99

Fig. 3. Scattering patterns of 43 endemic and external spinach accessions.

CONCLUSIONS

Results of this investigation approved that there was recommended for spring planting in Isfahan conditions. diversity in traits of native spinach accessions and Among the native accessions of Iran, “Varamin88” is therefore, it is very valuable for the researchers. So that, the only optimum accession with thick leaf, desirable the Iranian accessions like “Varamin88” and “Varamin performance, and notable fresh and dry weight, which Advanced Prickly” have a better adaptation to the makes it suitable for mechanized cultivation and is weather conditions of Isfahan and have shown favorable adapted to the spring in Isfahan. Generally, “Varamin88” traits such as yield, fresh and dry weight, petiole size, is recommended for planting in Isfahan between studied petiole attitude, and leaf number. Native accessions of accessions. “Hamedan2”, “Varamin88” and “Varamin Prickly” with the longest growing to flowering period are

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